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. 2020 Apr 21;14:246. doi: 10.3389/fnins.2020.00246

FIGURE 5.

FIGURE 5

Exosomes impede the uptake of preformed fibrils (PFFs) by neuronal cultures. (A) Primary mouse neuronal cultures (5div) were treated with either free or exosome-bound PFFs for 8 and 24 h. Following trypsinization, cell lysates were extracted with Triton X-100 and analyzed for the presence of α-syn species by western blotting using the specific α-syn antibody C-20. Increased α-syn high-molecular-weight species were evident in PFF-treated primary cultures as early as 8 h and significantly increased at 24 h. Graph shows the densitometric quantification from three independent experiments. Statistical analysis was with Student’s t-test. (B) Immunofluorescent confocal images of mouse primary cortical neurons treated for 48 h with free or exosome-bound PFFs following staining with Tuj-1 and C-20 antibodies. 3D reconstruction analysis revealed the differential localization of free PFFs versus exosome-bound PFFs in neuronal cultures. Imaris software was used for image processing. Scale bar represents 5 μm. (C) PFFs were incubated in vitro in the presence or absence of exosomes for up to 24 h and analyzed by western blotting with the C-20 and Syn-1 antibodies. Figure shows the gradual formation of higher-molecular-weight α-syn species (gel excluded) in the presence of exosomes. α-Syn (Syn-1) antibody further detected the presence of cleaved α-syn products below 15 kDa. ***p < 0.001.